Enzyme and protein active sites containing two or more interacting metal centers now figure prominently in metallobiochemistry. Systems containing metal clusters composed of Fe atoms (e.g., ferredoxins, sulfite reductase, hemerythrin, tyrosinase, ceruloplasmin, cytochrome oxidase), Mn atoms (photosystem II, catalase), and more than one element, or heteropolynuclear clusters (e.g., nitrogenase-Mo,Fe; cytochrome oxidase-Fe,Cu, hydrogenase and carbon monoxide dehydrogenase- Ni,Fe) have been identified. The ultimate goals of this project are to elucidate the structures of the metal clusters in metalloproteins, to determine the roles these clusters serve in the function of the protein , and to understand how nature designs a cluster for a specific purpose. This knowledge will provide a detailed understanding of biological processes and will aid in the design of pharmaceutical enzyme inhibitors and catalysts for various reactions. During the current report period, we completed studies of the structures of the Ni sites in eleven forms of Chromatium vinosum hydrogenase. These studies lead to three important conclusions related to the enzyme structure and function. First, the Ni-S distances differ by at most ~ 0.15 E, rather than 0.4 E as suggested by crystallographic information. Second, there is an oscillation in the edge energy observed in samples from enzyme in a ready conformation that indicates that the active site has two redox states (oxidized and reduced) and that these states differ by one electron. Last, there is a change in the Ni-Fe distance in the active site from a long distance (ca. 2.8 E) to a short distance (2.5 E) that is correlated with the reduction of the active site. This may be due to the binding of substrate or the loss of a solvent-derived O-donor bridging ligand upon reduction.